1. Field of the Invention
The present invention relates to in-vitro culturing of purified human leukocytes, and in particular, the present invention relates to a method of culturing of purified human leukocytes in a perfusion culturing system.
2. Description of the Prior Art
Attempts to manipulate the immune system with adoptive immunotherapy or by other means has had a long history. A very large number of experimental protocols have been tried in which agents thought to enhance and/or increase immunity have been given to patients. Most of these trials have not been successful and in the few cases in which success has been reported, it has been difficult to reproduce the successful aspects of the trial.
Adoptive immunotherapy in the contents of the present application involves the administration of immunologically active (immunocompetent) cells to an individual. These immunocompetent cells are taken either from the individual to be treated or from another individual. The purpose of administering immunocompetent cells to an individual is to provide a beneficial effect to the patient. For example, in the case of a cancer, cells are provided for the purpose of regressing and/or destroying a cancerous tumor.
Adoptive immunotherapy has been attempted by transferring immunocompetent cells from healthy animals to animals with a cancerous tumor. As such, animal experiments have suggested that an anti-tumor effect can be obtained with a high degree of antigen-specificity in certain tumor models. It has been found that the anti-tumor effect has been limited to certain tumors; and given the antigens-specificity of the effect, it has been assumed that in those cases (where antibodies have been ruled out as the effect or an important media of the effect) that leukocytes which include lymphocytes were involved.
More recently, these leukocytes have been described with reference to their anti-tumor activity and have been referred to as natural killer (NK) and lymphokine-activated killer (LAK) cells. (Other cells of the immune system which may be active in varying degrees regarding anti-tumor immunity also include cytotoxic T lymphocytes (CTL).)
NK and LAK cells are part of the immune system which preferentially lyse and/or kill target cells, including virally-infected and tumor cells. Rosenberg et al have shown in animal models, as well as in man, that lymphocytes obtained from peripheral blood in man or spleen in mouse can be activated within and for a very few days with recombinant interleukin-2 (rIL-2), a factor that activates certain lymphocytes such as LAK cells. Rosenberg has shown that LAK cells will have a regressive effect on tumors both in-vitro and in-vivo. This methodology has been applied to the treatment of cancer in man and encouraging results have been obtained in a significant number of patients, especially those with hypernephroma, melanoma and tumors of the colon.
However, a major difficulty in the protocol of Rosenberg et al has been the growth of the number of cells required to obtain a therapeutic effect. To obtain a therapeutic effect in the patients where a regressive effect on the tumor has resulted has been between 1.times.10.sup.10 and 2.times.10.sup.11 LAK cells. Since cells in regular tissue culture (static culture) can only be grown at a maximum of approximately 1 million cells/ml, the amount of tissue culture medium, flasks, incubators and the like needed for the growth of these cells has been enormous. Further, the manipulation of the cells in terms of feeding, removal of waste from the medium and harvesting has been highly labor-intensive. This problem has limited the number of cell preparations that can be readied for treatment of patients and potentially limits the number of treatment centers that could provide such treatment to patients. In addition, the problem of culturine a sufficient number of cells is further intensified if a larger amount of cells would provide a more beneficial treatment and produce better results in patients.